DNA encoding phoH polypeptides

ABSTRACT

The invention provides phoH homolog polypeptides and DNA (RNA) encoding phoH homolog polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing phoH homolog polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims benefit of provisional patent application Ser.No. 60/011,888, filed Feb. 20, 1996 and this application is acontinuation in part of PCT Application Number PCT/US97/02318, filedFeb. 19, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the phosphate inducible phoH family, hereinafterreferred to as "phoH homolog".

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. Staphylococcus aureus is the secondleading cause of bacteremia in cancer patients. Osteomyelitis, septicarthritis, septic thrombophlebitis and acute bacterial endocarditis arealso relatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past 20 years. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains which are resistant to some or all of thestandard antibiotics. This has created a demand for both newanti-microbial agents and diagnostic tests for this organism.

The phoH genes are phosphate starvation inducible in bacteria. Theencoded protein in Escherichia coli has been shown to contain nucleotidebinding domains, in particular for ATP binding (see Kim S K, Makino K,Amemura M, Shinagawa H, Nakata A. J. Bacteriol., March 1993;175(5):1316-1324). Such genes would play an important role in theadaptation to low phosphate concentrations, such as occurs duringinfection, and as such are targets for antibacterial strategies.

Substantial effort has been invested this century in the successfuldiscovery and development of antibacterials. Paradoxically althoughantibacterials are devised to eradicate infection in mammals we knowalmost nothing of the physiology of bacterial pathogens in infectivesituations in the host. Using sequences from the Staphylococcus aureuschromosome, we have developed an RT-PCR based procedure which allows usto identify those bacterial genes transcribed at any stage of infectionand also from different niches of infection. The derivation of suchinformation is a critical first step in understanding the globalresponse of the bacterial gene complement to the host environment. Fromthe knowledge of bacterial genes both of known and unknown functionwhich are widely transcribed in the host it is possible to attempt toascertain by database searching those which are present only in theeubacteria. Further prioritization of such genes by consideration of thelikely role of their products towards the maintenance of infection andthe facility of setting up a screen for inhibitors of the biochemicalfunction indicated by their homology to characterised genes allows thecompilation of a shortlist for gene essentiality studies using geneticdeletion or controlled regulation techniques. The proteins expressed bygenes shown to be necessary for growth in vitro or in pathogenesis inanimal models provide novel targets for antibacterial screening to findagents which are broadly inhibitory towards pathogenesis. This inventionprovides S. aureus WCUH 29 polynucleotides which are transcribed ininfected tissue, in particular in both acute and chronic infections.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown PHOH₋₋ BACSU protein (SWISS-PROT: locus PHOH₋₋ BACSU, accessionP46343).

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel phoH homolog polypeptides by homology between theamino acid sequence set out in Table 1 [SEQ ID NO: 2] and a known aminoacid sequence or sequences of other proteins such as PHOH₋₋ BACSUprotein.

It is a further object of the invention to provide polynucleotides thatencode phoH homolog polypeptides, particularly polynucleotides thatencode the polypeptide herein designated phoH homolog.

In a particularly preferred embodiment of the invention, thepolynucleotide comprises a region encoding phoH homolog polypeptidescomprising the sequence set out in Table 1 [SEQ ID NO:1] which includesa full length gene, or a variant thereof.

In another particularly preferred embodiment of the invention, there isa novel phoH homolog protein from Staphylococcus aureus comprising theamino acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.

In accordance with another aspect of the invention, there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Staphylococcus aureus WCUH 29 strain contained in the depositedstrain.

As a further aspect of the invention, there are provided isolatednucleic acid molecules encoding phoH homolog, particularlyStaphylococcus aureus phoH homolog, including mRNAs, cDNAs, genomicDNAs. Further embodiments of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulvariants thereof, and compositions comprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of phoH homolog and polypeptides encodedthereby.

As another aspect of the invention, there are provided novelpolypeptides of Staphylococcus aureus referred to herein as phoH homologas well as biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of phoH homolog polypeptide encoded by naturally occurringalleles of the phoH homolog gene.

In a preferred embodiment of the invention, there are provided methodsfor producing the aforementioned phoH homolog polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing phoHhomolog expression, treating disease, for example, disease, such as,infections of the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), assaying genetic variation, and administeringa phoH homolog polypeptide or polynucleotide to an organism to raise animmunological response against a bacteria, especially Staphylococcusaureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention, there are provided polynucleotides thathybridize to phoH homolog polynucleotide sequences, particularly understringent conditions.

In certain preferred embodiments of the invention, there are providedantibodies against phoH homolog polypeptides.

In other embodiments of the invention, there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided phoH homolog agonists and antagonists, preferablybacteriostatic or bactericidal agonists and antagonists.

In a further aspect of the invention, there are provided compositionscomprising a phoH homolog polynucleotide or a phoH homolog polypeptidefor administration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Host cell" is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, "identity" also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. "Identity" and "similarity" can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% "identity" to areference nucleotide sequence of SEQ ID NO: 1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO: 1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5' or 3' terminal positions ofthe reference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel phoH homolog polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novel phoHhomolog of Staphylococcus aureus, which is related by amino acidsequence homology to PHOH₋₋ BACSU polypeptide. The invention relatesespecially to phoH homolog having the nucleotide and amino acidsequences set out in Table 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2]respectively, and to the phoH homolog nucleotide sequences of the DNA inthe deposited strain and amino acid sequences encoded thereby.

                                      TABLE 1                                     __________________________________________________________________________    phoH homolog Polynucleotide and Polypeptide Sequences                         __________________________________________________________________________    (A) Sequences from Staphylococcus aureus phoH homolog polynucleotide          sequence [SEQ                                                                 ID NO:1].                                                                     5'-1  ATGAAAAGGA GCGCGTGTAT GCCTGGAATT ATACAAATAG                             ACGATATGAA                                                                    51 CCAATCTCAA GCTTTAATTG GAAATAATGA TGAACATTTA                                AAAGCAATTG                                                                    101 AAGAGAGTTT CGATGTTGTC ATCCATGCAA GAGGACAAGA                               AGTTGCCGTT                                                                    151 AAAGGTACAA AATAGAAAA CGTAGAAAAA GCGGAATCAG                                TATTAATCAA                                                                    201 TTTGCTGAAG GTTATTGATT TAGGTAATAA TATTACAATT                               AAAGATGTTG                                                                    251 AAGCAGCTAT TAAAATGGCG CATAATAACA CAATTCAACA                               TCTGTTAGAT                                                                    301 TTATATGATG AAGAGATAAC TAAAGATGCA TTTGGTAAGA                               CGATTCGTGC                                                                    351 GAAAACGATG GGGCAACGTA TATATGTTAA TGCCATGAAA                               AATAATGATT                                                                    401 TAGTATTTGG TATAGGTCCT GCTGGTACAG GTAAGACATT                               CTTAGCTGTA                                                                    451 GTTTATGCAG CAAAGCAACT CCGTAAAGGT GCTCCTAAAC                               GTATTGTATT                                                                    501 AACAAGACCT GCTGTTGAAG CAGGAGAGTC ACTTGGATTT                               TTACCAGGAG                                                                    551 ATTTGAAAGA AAAGGTAGAT CCATATTTAA GACCTTTATA                               TGATGGTCTA                                                                    601 TATACTGTTC TTGGGCGTGA ACAAACAGAG CGATTTATTG                               AAAGAGGCAT                                                                    651 TATCGAAATA GCGCCACTTG CATATATGCG CGGACGAACA                               TTAGAAGATG                                                                    701 CATTTGTAAT TCTTGATGAG GCGCAGAATA CGACACATGC                               GCAAATGAAA                                                                    751 ATGTTTTTAA CAAGACTAGG TTTTGGCTCA AAAATGGTAG                               TTACTGGTGA                                                                    801 CCAAACTCAA ATCGATTTAC CTAAAGGTGT TAAAAGTGGA                               CTTAAGGAAG                                                                    851 CGGTCAGTAG GTTACACAAC GTTAAAGGTA TAAGTATATT                               GAAATTAGAT                                                                    901 CAGAGCGATG TAGTAAGACA TCCATTGGTA AGTAACATCA                               TTGAACATTA                                                                    951 TGAAGGAGAG AATTAA                                                         3'                                                                            (B) phoH homolog polypeptide sequence deduced from the polynucleotide         sequence in this                                                              table [SEQ ID NO: 2].                                                         NH.sub.2 -1  MKRSACMPGI IQIDDMNQSQ ALIGNNDEHL KAIEESFDVV                      IHARGQEVAV                                                                    51 KGTKIENVEK AESVLINLLK VIDLGNNITI KDVEAAIKMA                                HNNTIQHLLD                                                                    101 LYDEEITKDA FGKTIRAKTM GQRIYVNAMK NNDLVFGIGP                               AGTGKTFLAV                                                                    151 VYAAKQLRKG AGKRIVLTRP AVEAGESLFG LPGDLKEKVD                               PYLRPLYDGL                                                                    201 YTVLGREQTE RFIERGIIEI APLAYMRGRT LEDAFVILDE                               AQNTTHAQMK                                                                    251 MFLTRLGFGS KMVVTGDQTG IDLPKGVKSG LKEAVSRLHN                               VKGISILKLD                                                                    301 QSDVVRGPLV SKIIEHYEGE N*--COOH                                            (C) Polynucleotide sequence embodiments [SEQ ID NO: 1].                       X-(R.sub.1).sub.n -1 ATGAAAAGGA GCGCGTGTAT GCCTGGAATT ATACAAATAG              ACGATATGAA                                                                    51 CCAATCTCAA GCTTTAATTG GAAATAATGA TGAACATTTA                                AAAGCAATTG                                                                    101 AAGAGAGTTT CGATGTTGTC ATCCATGCAA GAGGAACAAGA                              AGTTGCCGTT                                                                    151 AAAGGTACAA AAATAGAAAA CGTAGAAAAA GCGGAATCAG                               TATTAATCAA                                                                    201 TTTGCTGAAG GTTATTGATT TAGGTAATAA TATTACAATT                               AAAGATGTTG                                                                    251 AAGCAGCTAT TAAAATGGCG CATAATAACA CAATTCAACA                               TCTGTTAGAT                                                                    301 TTATATGATG AAGAGATAAC TAAAGATGCA TTTGGTAAGA                               CGATTCGTGC                                                                    351 GAAAACGATG GGGCAACGTA TATATGTTAA TGCCATGAAA                               AATAATGATT                                                                    401 TAGTATTTGG TATAGGTCCT GCTGGTACAG GTAAGACATT                               CTTAGCTGTA                                                                    451 GTTTATGCAG CAAAGCAACT CCGTAAAGGT GCTGGTAAAC                               GTATTGTATT                                                                    501 AACAAGACCT GCTGTTGAAG CAGGAGAGTC ACTTGGATTT                               TTACCAGGAG                                                                    551 ATTTGAAAGA AAAGGTAGAT CCATATTTAA GACCTTTATA                               TGATGGTCTA                                                                    601 TATACTGTTC TTGGGCGTGA ACAAACAGAG CGATTTATTG                               AAAGAGGCAT                                                                    651 TATCGAAATA GCGCCACTTG CATATATGCG CGGACGAACA                               TTAGAAGATG                                                                    701 CATTTGTAAT TCTTGATGAG GCGCAGAATA CGACACATGC                               GCAAATGAAA                                                                    751 ATGTTTTTAA CAAGACTAGG TTTTGGCTCA AAAATGGTAG                               TTACTGGTGA                                                                    801 CCAAACTCAA ATCGATTTAC CTAAAGGTGT TAAAAGTGGA                               CTTAAGGAAG                                                                    851 CGGTCAGTAG GTTACACAAC GTTAAAGGTA TAAGTATATT                               GAAATTAGAT                                                                    901 CAGAGCGATG TAGTAAGACA TCCATTGGTA AGTAAGATCA                               TTGAACATTA                                                                    951 TGAAGGAGAG AATTAA                                                         (R.sub.2).sub.n -Y                                                            (D) Polypeptide sequence embodiments [SEQ ID NO:2 ].                          X-(R.sub.i).sub.n.sup.-1 MKRSACMPGI IQIDDMNQSQ ALIGNNDEHL KAIEESFDVV          IHARGQEVAV                                                                    51 KGTKIENVEK AESVLINLLK VIDLGNNITI KDVEAAIKMA                                HNNTIQHLLD                                                                    101 LYDEEITKDA FGKTIRAKTM GQRIYVNAMK NNDLVFGIGP                               AGTGKTFLAV                                                                    151 VYAAKQLRKG AGKRIVLTRP AVEAGESLGF LPGKLKEKVD                               PYLRPLYDGL                                                                    201 YTVLGREQTE RFIERGIIEI APLAYMRGRT LEDAFVILDE                               AQNTTHAQMK                                                                    251 MFLTRLGFGS KMVVTGDQTG IDLPKGVKSG LKEAVSRLHN                               VKGISILKLD                                                                    301 QSDVVRHPLV SKIIEHYEGE N*-(R.sub.2).sub.n -Y                               (E) Sequences from Staphylococcus aureus phoH homolog polynucleotide ORF      sequence                                                                      ]SEQ ID NO:3].                                                                5'-ATGAAAAGGA GCGCGTGTAT GCCTGGAATT ATACAAATAG ACGATATGAA                     51 CCAATCTCAA GCTTTAATTG GAAATAATGA TGAACATTTA                                AAAGCAATTG                                                                    101 AAGAGAGTTT CGATGTTGTC ATCCATGCAA GAGGACAAGA                               AGTTGCCGTT                                                                    151 AAAGGTACAA AAATAGAAAA CGTAGAAAAA GCGGAATCAG                               TATTAATCAA                                                                    201 TTTGCTGAAG GTTATTGATT TAGGTAATAA TATTACAATT                               AAAGATCTTG                                                                    251 AAGCAGCTAT TAAAATGGCG CATAATAACA  CAATTCAACA                              TCTGTTAGAT                                                                    301 TTATATGATG AAGAGATAAC TAAAGATGCA TTTGGTAAGA                               CGATTCGTGC                                                                    351 GAAAACGATG GGGCAACGTA TATATGTTAA TGCCATGAAA                               AATAATGATT                                                                    401 TAGTATTTGG TATAGGTCCT GCTGGTACAG GTAAGACATT                               CTTAGCTGTA                                                                    451 GTTTATGCAG CAAAGCAACT CCGTAAAGGT GCTGGTAAAC                               GTATTGTATT                                                                    501 AACAAGACCT GCTGTTGAAG CAGGAGAGTC ACTTGGATTT                               TTACCAGGAG                                                                    551 ATTTGAAAGA AAAGGTAGAT CCATATTTAA GACCTTTATA                               TGATGGTCTA                                                                    601 TATACTGTTC TTGGGCGTGA ACAAACAGAG CGATTTATTG                               AAAGAGGCAT                                                                    651 TATCGAAATA GCGCCACTTG CATATATGCG CGGACGAACA                               TTAGAAGATG                                                                    701 CATTTGTAAT TCTTGATGAG GCGCAGAATA CGACACATGC                               GCAAATGAAA                                                                    751 ATGTTTTTAA CAAGACTAGG TTTTGGCTCA AAAATGGTAG                               TTACTGGTGA                                                                    801 CCAAACTCAA ATCGATTTAC CTAAAGGTGT TAAAAGTGGA                               CTTAAGGAAG                                                                    851 CGGTCAGTAG GTTACACAAC GTTAAAGGTA TAAGTATATT                               GAAATTAGAT                                                                    901 CAGAGCGATG TAGTAAGACA TCCATTGGTA AGTAAGATCA                               TTGAACATTA                                                                    951 TGAAGGAGAG AAT-3'                                                         (F) phoH homolog poypeptide sequence deduced from the polynucleotide ORF      sequence in                                                                   this table [SEQ ID NO:4].                                                     NH.sub.2 -MKRSACMPGI IQIDDMNQSQ ALIGNNDEHL KAIEESFDVV IHARGQEVAV              51 KGTKIENVEK  AESVLINLLK VIDLGNNITI KDVEAAIKMA                               HNNTIQHLLD                                                                    101 LYDEEITKDA FGKTIRAKTM GQRIYVNAMK NNDLVFGIGP                               AGTGKTFLAV                                                                    151 VYAAKQLRKG AGKRIVLTRP AVEAGESLGF LPGDLKEKVD                               PYLRPLYDGL                                                                    201 YTVLGREQTE RFIERGIIEI APLAYMRGRT LEDAFVILDE                               AQNTTHAQMK                                                                    251 MFLTRLGFGS KMVVTGDQTQ IDLPKGVKSG LKEAVSRLHN                               VKGISILKLD                                                                    301 QSKVVRHPLV SKIIEHYEGE N--cooh                                             __________________________________________________________________________     Deposited materials                                                      

Deposited materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, and isreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as "thedeposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length phoH homolog gene. Thesequence of the polynucleotides contained in the deposited strain, aswell as the amino acid sequence of the polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of Table 1[SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of phoH homolog, and also those which have at least 70%identity to a polypeptide of Table 1 [SEQ ID NOS:2 and 4] or therelevant portion, preferably at least 80% identity to a polypeptide ofTable 1 [SEQ ID NOS:2 and 4], and more preferably at least 90%similarity (more preferably at least 90% identity) to a polypeptide ofTable 1 [SEQ ID NOS:2 and 4] and still more preferably at least 95%similarity (still more preferably at least 95% identity) to apolypeptide of Table 1 [SEQ ID NOS:2 and 4] and also include portions ofsuch polypeptides with such portion of the polypeptide generallycontaining at least 30 amino acids and more preferably at least 50 aminoacids.

The invention also includes polypeptides of the formula set forth inTable 1 (D) [SEQ ID NO:2] wherein, at the amino terminus, X is hydrogen,and at the carboxyl terminus, Y is hydrogen or a metal, R₁ and R₂ is anyamino acid residue, and n is an integer between 1 and 1000. Any stretchof amino acid residues denoted by either R group, where R is greaterthan 1, may be either a heteropolymer or a homopolymer, preferably aheteropolymer.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with phoH homolog polypeptidesfragments may be "free-standing," or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NOS:2 and 4], orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Staphylococcus aureus, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of phoH homolog, including those witha similar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Staphylococcusaureus or the ability to initiate, or maintain cause disease in anindividual, particularly a human.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotides,including the full length gene, that encode the phoH homolog polypeptidehaving a deduced amino acid sequence of Table 1 [SEQ ID NOS:2 and 4] andpolynucleotides closely related thereto and variants thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NOS:1 and 3], a polynucleotide of theinvention encoding phoH homolog polypeptide may be obtained usingstandard cloning and screening methods, such as those for cloning andsequencing chromosomal DNA fragments from bacteria using Staphylococcusaureus WCUH 29 cells as starting material, followed by obtaining a fulllength clone. For example, to obtain a polynucleotide sequence of theinvention, such as a sequence given in Table 1 [SEQ ID NOS:1 and 3],typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently, such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Illustrative of the invention, the polynucleotide setout in Table 1 [SEQ ID NO: 1] was discovered in a DNA library derivedfrom Staphylococcus aureus WCUH 29.

The DNA sequence set out in Table 1 [SEQ ID NO: 1] contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in Table 1 [SEQ ID NO:2] with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The polynucleotide of SEQ ID NO: 1,between nucleotide number 1 through number 963 encodes the polypeptideof SEQ ID NO:2. The stop codon begins at nucleotide number 966 of SEQ IDNO: 1.

The phoH homolog polypeptide of the invention is structurally related toother proteins of the phosphate inducible phoH family, as shown by theresults of sequencing the DNA encoding phoH homolog of the depositedstrain. The protein exhibits greatest homology to PHOH₋₋ BACSU proteinamong known proteins (SWISS-PROT: locus PHOH₋₋ BACSU, accession P46343).The phoH homolog of Table 1 [SEQ ID NO:2] has about 68% identity overits entire length and about 81% similarity over its entire length withthe amino acid sequence of PHOH₋₋ BACSU polypeptide.

The invention provides a polynucleotide sequence identical over itsentire length to the coding sequence in Table 1 [SEQ ID NO: 1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro-protein sequence. The polynucleotidemay also contain non-coding sequences, including for example, but notlimited to non-coding 5' and 3' sequences, such as the transcribed,non-translated sequences, termination signals, ribosome binding sites,sequences that stabilize mRNA, introns, polyadenylation signals, andadditional coding sequence which encode additional amino acids. Forexample, a marker sequence that facilitates purification of the fusedpolypeptide can be encoded. In certain embodiments of the invention, themarker sequence is a hexa-histidine peptide, as provided in the pQEvector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad.Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al., Cell 37: 767(1984). Polynucleotides of the invention also include, but are notlimited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofcomprising nucleotide 1 to 963 or 966 set forth in SEQ ID NO: 1 of Table1 which encode the phoH homolog polypeptide.

The invention also includes polynucleotides of the formula set forth inTable 1 (C)[SEQ ID NO: 1] wherein, at the 5' end of the molecule, X ishydrogen, and at the 3' end of the molecule, Y is hydrogen or a metal,R₁ and R₂ is any nucleic acid residue, and n is an integer between 1 and1000. Any stretch of nucleic acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus phoHhomolog having the amino acid sequence set out in Table 1 [SEQ ID NO:2].The term also encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, interrupted by integrated phage or an insertion sequence orediting) together with additional regions, that also may contain codingand/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingphoH homolog variants, that have the amino acid sequence of phoH homologpolypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of phoH homolog.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding phoH homolog polypeptide having an amino acid sequence set outin Table 1 [SEQ ID NOS:2 and 4], and polynucleotides that arecomplementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least80% identical over its entire length to a polynucleotide encoding phoHhomolog polypeptide of the deposited strain and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical over their entire length to the same are particularlypreferred, and among these particularly preferred polynucleotides, thosewith at least 95% are especially preferred. Furthermore, those with atleast 97% are highly preferred among those with at least 95%, and amongthese those with at least 98% and at least 99% are particularly highlypreferred, with at least 99% being the more preferred.

Preferred embodiments are polynucleotides that encode polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by the DNA of Table 1 [SEQ ID NO: 1].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms "stringent conditions" and "stringent hybridizationconditions" mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at about 65° C. Hybridization and wash conditions are well known andexemplified in Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter11 therein.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO: 1 or SEQ ID NO: 3 under stringent hybridization conditionswith a probe having the sequence of said polynucleotide sequence setforth in SEQ ID NO: 1 or a fragment thereof; and isolating said DNAsequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding phoHhomolog and to isolate cDNA and genomic clones of other genes that havea high sequence similarity to the phoH homolog gene. Such probesgenerally will comprise at least 15 bases. Preferably, such probes willhave at least 30 bases and may have at least 50 bases. Particularlypreferred probes will have at least 30 bases and will have 50 bases orless.

For example, the coding region of the phoH homolog gene may be isolatedby screening using the DNA sequence provided in SEQ ID NO: 1 tosynthesize an oligonucleotide probe. A labeled oligonucleotide having asequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The polynucleotides and polypeptides of the invention may be employed,for example, as :research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS:1 and/or 2 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, enterococci E. coli, streptomycesand Bacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoplera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the phoH homologpolynucleotides of the invention for use as diagnostic reagents.Detection of phoH homolog in a eukaryote, particularly a mammal, andespecially a human, will provide a diagnostic method for diagnosis of adisease. Eukaryotes (herein also "individual(s)"), particularly mammals,and especially humans, infected with an organism comprising the phoHhomolog gene may be detected at the nucleic acid level by a variety oftechniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled phoH homolog polynucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations in theelectrophoretic mobility of the DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,Science, 230: 1242 (1985). Sequence changes at specific locations alsomay be revealed by nuclease protection assays, such as RNase and S1protection or a chemical cleavage method. See, e.g., Cotton et al.,Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to a nucleic acidencoding phoH homolog can be used to identify and analyze mutations.Examples of representative primers are shown below in Table 2.

                  TABLE 2                                                         ______________________________________                                        Primers for amplification of phoH homolog polynucleotides                     SEQ ID NO   PRIMER SEQUENCE                                                   ______________________________________                                        5           35'-GCTTTAATTG GAAATAATGA TG-3'                                   6           5'-ATACTTATACCTTTAACGTTG-3'                                       ______________________________________                                    

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying phoH homolog DNA isolated froma sample derived from an individual. The primers may be used to amplifythe gene isolated from an infected individual such that the gene maythen be subject to various techniques for elucidation of the DNAsequence. In this way, mutations in the DNA sequence may be detected andused to diagnose infection and to serotype and/or classify theinfectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and perinephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of Table 1 [SEQ ID NO: 1]. Increased or decreasedexpression of phoH homolog polynucleotide can be measured using any onof the methods well known in the art for the quantitation ofpolynucleotides, such as, for example, amplification, PCR, RT-PCR, RNaseprotection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of phoH homolog protein compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a phoH homolog protein, in a sample derived from a host arewell-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. "Antibodies" as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-phoH homolog or from naive libraries(McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al.,(1992) Biotechnology 10, 779-783). The affinity of these antibodies canalso be improved by chain shuffling (Clackson, T. et al., (1991) Nature352, 624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope--termed `bispecific` antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against phoH homolog-polypeptide may beemployed to treat infections, particularly bacterial infections andespecially disease, such as, infections of the upper respiratory tract(e.g., otitis media, bacterial tracheitis, acute epiglottitis,thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac(e.g., infective endocarditis), gastrointestinal (e.g., secretorydiarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebralabscess), eye (e.g., blepharitis, conjunctivitis, keratitis,endophthalmitis, preseptal and orbital cellulitis, darcryocystitis),kidney and urinary tract (e.g., epididymitis, intrarenal and perinephricabsces, toxic shock syndrome), skin (e.g., impetigo, folliculitis,cutaneous abscesses, cellulitis, wound infection, bacterial myositis)bone and joint (e.g., septic arthritis, osteomyelitis).

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term "antigenically equivalent derivative" as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm "immunologically equivalent derivative" as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized"; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(Tang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS USA 1984:81,5849).

Antagonists and agonists--assays and molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of phoHhomolog polypeptides or polynucleotides, particularly those compoundsthat are bacteriostatic and/or bactericidal. The method of screening mayinvolve high-throughput techniques. For example, to screen for agonistsor antagonists, a synthetic reaction mix, a cellular compartment, suchas a membrane, cell envelope or cell wall, or a preparation of anythereof, comprising phoH homolog polypeptide and a labeled substrate orligand of such polypeptide is incubated in the absence or the presenceof a candidate molecule that may be a phoH homolog agonist orantagonist. The ability of the candidate molecule to agonize orantagonize the phoH homolog polypeptide is reflected in decreasedbinding of the labeled ligand or decreased production of product fromsuch substrate. Molecules that bind gratuitously, i.e., without inducingthe effects of phoH homolog polypeptide are most likely to be goodantagonists. Molecules that bind well and increase the rate of productproduction from substrate are agonists. Detection of the rate or levelof production of product from substrate may be enhanced by using areporter system. Reporter systems that may be useful in this regardinclude but are not limited to colorimetric labeled substrate convertedinto product, a reporter gene that is responsive to changes in phoHhomolog polynucleotide or polypeptide activity, and binding assays knownin the art.

Another example of an assay for phoH homolog antagonists is acompetitive assay that combines phoH homolog and a potential antagonistwith phoH homolog-binding molecules, recombinant phoH homolog bindingmolecules, natural substrates or ligands, or substrate or ligandmimetics, under appropriate conditions for a competitive inhibitionassay. The phoH homolog can be labeled, such as by radioactivity or acolorimetric compound, such that the number of phoH homolog moleculesbound to a binding molecule or converted to product can be determinedaccurately to assess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing phoH homolog-induced activities, thereby preventing the actionof phoH homolog by excluding phoH homolog from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of phoH homolog.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock phoH homolog protein-mediated mammalian cell invasion by, forexample, initiating phosphorylation of mammalian tyrosine kinases(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block bacterialadhesion between mammalian extracellular matrix proteins and bacterialphoH homolog proteins that mediate tissue damage and; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with phoH homolog, or a fragment orvariant thereof, adequate to produce antibody and/or T cell immuneresponse to protect said individual from infection, particularlybacterial infection and most particularly Staphylococcus aureusinfection. Also provided are methods whereby such immunological responseslows bacterial replication. Yet another aspect of the invention relatesto a method of inducing immunological response in an individual whichcomprises delivering to such individual a nucleic acid vector to directexpression of phoH homolog, or a fragment or a variant thereof, forexpressing phoH homolog, or a fragment or a variant thereof in vivo inorder to induce an immunological response, such as, to produce antibodyand/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual from disease, whether that disease is already establishedwithin the individual or not. One way of administering the gene is byaccelerating it into the desired cells as a coating on particles orotherwise.

Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid,or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a phoH homolog or protein codedtherefrom, wherein the composition comprises a recombinant phoH homologor protein coded therefrom comprising DNA which codes for and expressesan antigen of said phoH homolog or protein coded therefrom. Theimmunological response may be used therapeutically or prophylacticallyand may take the form of antibody immunity or cellular immunity such asthat arising from CTL or CD4+ T cells.

A phoH homolog polypeptide or a fragment thereof may be fused withco-protein which may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae, Glutathione-S-transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the bodily fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certain phoHhomolog protein, it is to be understood that this covers fragments ofthe naturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain Selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO: 1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. The sequencing data from two or more clonescontaining overlapping Staphylococcus aureus DNAs was used to constructthe contiguous DNA sequence in SEQ ID NO: 1. Libraries may be preparedby routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bsh1235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2 The Determination of Expression during Infection of a Genefrom Staphylococcus Aureus

Necrotic fatty tissue from a 72 hour groin infection or an excisedkidney from an 8 day chronic kidney infection of Staphylococcus aureusWCUH29 in the mouse is efficiently disrupted and processed in thepresence of chaotropic agents and RNAase inhibitor to provide a mixtureof animal and bacterial RNA. The optimal conditions for disruption andprocessing to give stable preparations and high yields of bacterial RNAare followed by the use of hybridisation to a radiolabelledoligonucleotide specific to Staphylococcus aureus 16S RNA on Northernblots. The RNAase free, DNAase free, DNA and protein free preparationsof RNA obtained are suitable for Reverse Transcription PCR (RT-PCR)using unique primer pairs designed from the sequence of each gene ofStaphylococcus aureus WCUH29.

a) Isolation of tissue infected with Staphylococcus aureus WCUH29 from amouse animal model of infection (groin)

10 ml. volumes of sterile nutrient broth (No.2 Oxoid) are seeded withisolated, individual colonies of Staphylococcus aureus WCUH29 from anagar culture plate. The cultures are incubated aerobically (staticculture) at 37° C. for 16-20 hours. 4 week old mice (female,18 g-22 g,strain MF1) are each infected by subcutaneous injection of 0.5 ml. ofthis broth culture of Staphylococcus aureus WCUH29 (diluted in broth toapproximately 10⁸ cfu/ml.) into the anterior, right lower quadrant(groin area). Mice should be monitored regularly during the first 24hours after infection, then daily until termination of study. Animalswith signs of systemic infection, i.e. lethargy, ruffled appearance,isolation from group, should be monitored closely and if signs progressto moribundancy, the animal should be culled immediately.

Visible external signs of lesion development will be seen 24-48 h afterinfection. Examination of the abdomen of the animal will show the raisedoutline of the abscess beneath the skin. The localised lesion shouldremain in the right lower quadrant, but may occasionally spread to theleft lower quadrant, and superiorly to the thorax. On occasions, theabscess may rupture through the overlying skin layers. In such cases theaffected animal should be culled immediately and the tissues sampled ifpossible. Failure to cull the animal may result in the necrotic skintissue overlying the abscess being sloughed off, exposing the abdominalmuscle wall.

Approximately 96 hours after infection, animals are killed using carbondioxide asphyxiation. To minimise delay between death and tissueprocessing /storage, mice should be killed individually rather than ingroups. The dead animal is placed onto its back and the fur swabbedliberally with 70% alcohol. An initial incision using scissors is madethrough the skin of the abdominal left lower quadrant, travellingsuperiorly up to, then across the thorax. The incision is completed bycutting inferiorly to the abdominal lower right quadrant. Care should betaken not to penetrate the abdominal wall. Holding the skin flap withforceps, the skin is gently pulled way from the abdomen. The exposedabscess, which covers the peritoneal wall but generally does notpenetrate the muscle sheet completely, is excised, taking care not topuncture the viscera

The abscess/muscle sheet and other infected tissue may require cuttingin sections, prior to flash-freezing in liquid nitrogen, therebyallowing easier storage in plastic collecting vials.

b) Isolation of tissue infected with Staphylococcus aureus WCUH29 from amurine model of hematogenous pyelonephritis.

Overnight cultures of Staphylococcus aureus WCUH29 were started fromsingle colonies in 5 ml of tryptic soy broth (TSB) and grown at 37° C.with shaking. The cultures were then washed twice in sterilephosphate-buffered saline (PBS) and diluted to an A600=0.3. Male CD-1mice (18-20 g) were infected with 0.2 ml of this suspension by tail veininoculation using a 30 g needle attached to a tuberculin syringe. Eachmouse receives approximately 4×10⁷ bacteria in this fashion. Mice aremonitored daily for signs of illness, and usually within 48 hours showsigns of lethargy, ruffled fur, sluggishness; animals which appearmoribund are euthanized prior to the end of the experiment.

All animals are euthanized via carbon dioxide overdose seven dayspost-infection. The animal is placed on its back and swabbed withethanol, and then with RNAZap, and instruments are swabbed as well. Theabdominal cavity is opened and the kidneys aseptically removed, cut intofour pieces, and placed in cryovials which are immediately frozen inliquid nitrogen.

c) Isolation of Staphylococcus aureus WCUH29 RNA from infected tissuesamples

4-6 infected tissue samples(each approx 0.5-0.7 g) in 2 ml screw-captubes are removed from -80° C. storage into a dry ice ethanol bath. In amicrobiological safety cabinet the samples are disrupted individuallywhilst the remaining samples are kept cold in the dry ice ethanol bath.To disrupt the bacteria within the tissue sample 1 ml of TRIzol Reagent(Gibco BRL, Life Technologies) is added followed by enough 0.1 mmzirconia/silica beads to almost fill the tube, the lid is replacedtaking care not to get any beads into the screw thread so as to ensure agood seal and eliminate aerosol generation. The sample is thenhomogenised in a Mini-BeadBeater Type BX-4 (Biospec Products). Necroticfatty tissue isstrain treated for 100 seconds at 5000 rpm in order toachieve bacterial lysis. In vivo grown bacteria require longer treatmentthan in vitro grown Staphylococcus aureus Staphylococcus which aredisrupted by a 30 second bead-beat.

After bead-beating the tubes are chilled on ice before opening in afume-hood as heat generated during disruption may degrade the TRIzol andrelease cyanide.

200 microliters of chloroform is then added and the tubes shaken by handfor 15 seconds to ensure complete mixing. After 2-3 minutes at roomtemperature the tubes are spun down at 12,000× g, 4 ° C. for 15 minutesand RNA extraction is then continued according to the method given bythe manufacturers of TRIzol Reagent i.e.:--The aqueous phase, approx 0.6ml, is transferred to a sterile eppendorf tube and 0.5 ml of isopropanolis added. After 10 minutes at room temperature the samples are spun at12,000× g, 4° C. for 10 minutes. The supernatant is removed anddiscarded then the RNA pellet is washed with 1 ml 75% ethanol. A briefvortex is used to mix the sample before centrifuging at 7,500× g, 4° C.for 5 minutes. The ethanol is removed and the RNA pellet dried undervacuum for no more than 5 minutes. Samples are then resuspended byrepeated pipetting in 100 microliters of DEPC treated water, followed by5-10 minutes at 55° C. Finally, after at least 1 minute on ice, 200units of Rnasin (Promega) is added.

RNA preparations are stored at -80° C. for up to one month. For longerterm storage the RNA precipitate can be stored at the wash stage of theprotocol in 75% ethanol for at least one year at -20° C.

Quality of the RNA isolated is assessed by running samples on 1% agarosegels. 1× TBE gels stained with ethidium bromide are used to visualisetotal RNA yields. To demonstrate the isolation of bacterial RNA from theinfected tissue 1× MOPS, 2.2M formaldehyde gels are run and vacuumblotted to Hybond-N (Amersham). The blot is then hybridised with a ³² Plabelled oligonucletide probe specific to 16s rRNA of Staphylococcusaureus (K. Greisen, M. Loeffelholz, A. Purohit and D. Leong. J. Clin.(1994) Microbiol. 32 335-351 ). An oligonucleotide of the sequence:5'-gctcctaaaaggttactccaccggc-3' is used as a probe. The size of thehybridising band is compared to that of control RNA isolated from invitro grown Staphylococcus aureus WCUH29 in the Northern blot. Correctsized bacterial 16s rRNA bands can be detected in total RNA sampleswhich show extensive degradation of the mammalian RNA when visualised onTBE gels.

d) The removal of DNA from Staphylococcus aureus WCUH29-derived RNA

DNA was removed from 73 microliter samples of RNA by a 15 minutetreatment on ice with 3 units of DNAaseI, amplification grade (GibcoBRL, Life Technologies) in the buffer supplied with the addition of 200units of Rnasin (Promega) in a final volume of 90 microliters.

The DNAase was inactivated and removed by treatment with TRIzol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol. DNAase treated RNA was resuspended in 73 microliters of DEPCtreated water with the addition of Rnasin as described in Method 1.

e) The preparation of cDNA from RNA samples derived from infected tissue

10 microliter samples of DNAase treated RNA are reverse transcribedusing.a SuperScript Preamplification System for First Strand cDNASynthesis kit (Gibco BRL, Life Technologies) according to themanufacturers instructions. 1 nanogram of random hexamers is used toprime each reaction. Controls without the addition of SuperScriptIIreverse transcriptase are also run. Both +/-RT samples are treated withRNaseH before proceeding to the PCR reaction.

f) The use of PCR to determine the presence of a bacterial cDNA species

PCR reactions are set up on ice in 0.2 ml tubes by adding the followingcomponents: 45 microliters PCR SUPERMIX (Gibco BRL, Life Technologies);1 microliter 50 mM MgCl₂, to adjust final concentration to 2.5 mM; 1microliter PCR primers(optimally 18-25 basepairs in length and designedto possess similar annealing temperatures), each primer at 10 mM initialconcentration; and 2 microliters cDNA.

PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 asfollows: 5 minutes at 95 ° C., then 50 cycles of 30 seconds each at 94°C., 42° C. and 72° C. followed by 3 minutes at 72° C. and then a holdtemperature of 4° C. (the number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product and optimally 8-30cycles if an estimation of the starting quantity of cDNA from the RTreaction is to be made); 10 microliter aliquots are then run out on 1%1× TBE gels stained with ethidium bromide with PCR product, if present,sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5'endwith a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™ 377 Sequenccrusing GeneScan™ software as supplied by Perkin Elmer).

RTIPCR controls may include +/- reverse transcriptase reactions, 16srRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Staphylococcus aureus WCUH29 genomicsequences.

To test the efficiency of the primer pairs they are used in DNA PCR withStaphylococcus aureus WCUH29 total DNA. PCR reactions are set up and runas described above using approx. 1 microgram of DNA in place of the cDNAand 35 cycles of PCR.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR: 1. Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and 2. Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in -RT controls.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 6                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 966 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #CCAATCTCAA    60CGTGTAT GCCTGGAATT ATACAAATAG ACGATATGAA                     #CGATGTTGTC   120ATAATGA TGAACATTTA AAAGCAATTG AAGAGAGTTT                     #CGTAGAAAAA   180GACAAGA AGTTGCCGTT AAAGGTACAA AAATAGAAAA                     #TATTACAATT   240TAATCAA TTTGCTGAAG GTTATTGATT TAGGTAATAA                     #TCTGTTAGAT   300CAGCTAT TAAAATGGCG CATAATAACA CAATTCAACA                     #GAAAACGATG   360AGATAAC TAAAGATGCA TTTGGTAAGA CGATTCGTGC                     #TATAGGTCCT   420ATGTTAA TGCCATGAAA AATAATGATT TAGTATTTGG                     #CCGTAAAGGT   480AGACATT CTTAGCTGTA GTTTATGCAG CAAAGCAACT                     #ACTTGGATTT   540TTGTATT AACAAGACCT GCTGTTGAAG CAGGAGAGTC                     #TGATGGTCTA   600TGAAAGA AAAGGTAGAT CCATATTTAA GACCTTTATA                     #TATCGAAATA   660GGCGTGA ACAAACAGAG CGATTTATTG AAAGAGGCAT                     #TCTTGATGAG   720ATATGCG CGGACGAACA TTAGAAGATG CATTTGTAAT                     #TTTTGGCTCA   780CACATGC GCAAATGAAA ATGTTTTTAA CAAGACTAGG                     #TAAAAGTGGA   840CTGGTGA CCAAACTCAA ATCGATTTAC CTAAAGGTGT                     #GAAATTAGAT   900TCAGTAG GTTACACAAC GTTAAAGGTA TAAGTATATT                     #TGAAGGAGAG   960TAAGACA TCCATTGGTA AGTAAGATCA TTGAACATTA                     #          966                                                                - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 321 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #Ile Gln Ile Asp Asp Metys Met Pro Gly Ile                                    #                 15                                                          #Asp Glu His Leu Lys Alaeu Ile Gly Asn Asn                                    #             30                                                              #Ala Arg Gly Gln Glu Valsp Val Val Ile His                                    #         45                                                                  #Glu Lys Ala Glu Ser Valys Ile Glu Asn Val                                    #     60                                                                      #Gly Asn Asn Ile Thr Ileys Val Ile Asp Leu                                    # 80                                                                          #His Asn Asn Thr Ile Glnla Ile Lys Met Ala                                    #                 95                                                          #Thr Lys Asp Ala Phe Glyyr Asp Glu Glu Ile                                    #            110                                                              #Arg Ile Tyr Val Asn Alays Thr Met Gly Gln                                    #        125                                                                  #Gly Pro Ala Gly Thr Glyeu Val Phe Gly Ile                                    #    140                                                                      #Lys Gln Leu Arg Lys Glyal Val Tyr Ala Ala                                    #160                                                                          #Ala Val Glu Ala Gly Glual Leu Thr Arg Pro                                    #                175                                                          #Glu Lys Val Asp Pro Tyrro Gly Asp Leu Lys                                    #            190                                                              #Val Leu Gly Arg Glu Glnsp Gly Leu Tyr Thr                                    #        205                                                                  #Glu Ile Ala Pro Leu Alalu Arg Gly Ile Ile                                    #    220                                                                      #Phe Val Ile Leu Asp Gluhr Leu Glu Asp Ala                                    #240                                                                          #Met Phe Leu Thr Arg Leuis Ala Gln Met Lys                                    #                255                                                          #Asp Gln Thr Gln Ile Aspet Val Val Thr Gly                                    #            270                                                              #Glu Ala Val Ser Arg Leuys Ser Gly Leu Lys                                    #        285                                                                  #Leu Asp Gln Ser Asp Valle Ser Ile Leu Lys                                    #    300                                                                      #Glu His Tyr Glu Gly Glual Ser Lys Ile Ile                                    #320                                                                          -  Asn                                                                        - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 963 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #CCAATCTCAA    60CGTGTAT GCCTGGAATT ATACAAATAG ACGATATGAA                     #CGATGTTGTC   120ATAATGA TGAACATTTA AAAGCAATTG AAGAGAGTTT                     #CGTAGAAAAA   180GACAAGA AGTTGCCGTT AAAGGTACAA AAATAGAAAA                     #TATTACAATT   240TAATCAA TTTGCTGAAG GTTATTGATT TAGGTAATAA                     #TCTGTTAGAT   300CAGCTAT TAAAATGGCG CATAATAACA CAATTCAACA                     #GAAAACGATG   360AGATAAC TAAAGATGCA TTTGGTAAGA CGATTCGTGC                     #TATAGGTCCT   420ATGTTAA TGCCATGAAA AATAATGATT TAGTATTTGG                     #CCGTAAAGGT   480AGACATT CTTAGCTGTA GTTTATGCAG CAAAGCAACT                     #ACTTGGATTT   540TTGTATT AACAAGACCT GCTGTTGAAG CAGGAGAGTC                     #TGATGGTCTA   600TGAAAGA AAAGGTAGAT CCATATTTAA GACCTTTATA                     #TATCGAAATA   660GGCGTGA ACAAACAGAG CGATTTATTG AAAGAGGCAT                     #TCTTGATGAG   720ATATGCG CGGACGAACA TTAGAAGATG CATTTGTAAT                     #TTTTGGCTCA   780CACATGC GCAAATGAAA ATGTTTTTAA CAAGACTAGG                     #TAAAAGTGGA   840CTGGTGA CCAAACTCAA ATCGATTTAC CTAAAGGTGT                     #GAAATTAGAT   900TCAGTAG GTTACACAAC GTTAAAGGTA TAAGTATATT                     #TGAAGGAGAG   960TAAGACA TCCATTGGTA AGTAAGATCA TTGAACATTA                     #            963                                                              - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 321 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #Ile Gln Ile Asp Asp Metys Met Pro Gly Ile                                    #                 15                                                          #Asp Glu His Leu Lys Alaeu Ile Gly Asn Asn                                    #             30                                                              #Ala Arg Gly Gln Glu Valsp Val Val Ile His                                    #         45                                                                  #Glu Lys Ala Glu Ser Valys Ile Glu Asn Val                                    #     60                                                                      #Gly Asn Asn Ile Thr Ileys Val Ile Asp Leu                                    # 80                                                                          #His Asn Asn Thr Ile Glnla Ile Lys Met Ala                                    #                 95                                                          #Thr Lys Asp Ala Phe Glyyr Asp Glu Glu Ile                                    #            110                                                              #Arg Ile Tyr Val Asn Alays Thr Met Gly Gln                                    #        125                                                                  #Gly Pro Ala Gly Thr Glyeu Val Phe Gly Ile                                    #    140                                                                      #Lys Gln Leu Arg Lys Glyal Val Tyr Ala Ala                                    #160                                                                          #Ala Val Glu Ala Gly Glual Leu Thr Arg Pro                                    #                175                                                          #Glu Lys Val Asp Pro Tyrro Gly Asp Leu Lys                                    #            190                                                              #Val Leu Gly Arg Glu Glnsp Gly Leu Tyr Thr                                    #        205                                                                  #Glu Ile Ala Pro Leu Alalu Arg Gly Ile Ile                                    #    220                                                                      #Phe Val Ile Leu Asp Gluhr Leu Glu Asp Ala                                    #240                                                                          #Met Phe Leu Thr Arg Leuis Ala Gln Met Lys                                    #                255                                                          #Asp Gln Thr Gln Ile Aspet Val Val Thr Gly                                    #            270                                                              #Glu Ala Val Ser Arg Leuys Ser Gly Leu Lys                                    #        285                                                                  #Leu Asp Gln Ser Asp Valle Ser Ile Leu Lys                                    #    300                                                                      #Glu His Tyr Glu Gly Glual Ser Lys Ile Ile                                    #320                                                                          -  Asn                                                                        - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #                 22ATGA TG                                                   - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #21                ACGTT G                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment encoding SEQID NO:
 2. 2. An isolated nucleic acid segment comprising a nucleotidesequence which is fully complementary to the polynucleotide of claim 1.3. An isolated vector comprising the polynucleotide segment of claim 1.4. An isolated vector comprising the nucleic acid segment of claim
 2. 5.An isolated host cell comprising the vector of claim
 3. 6. An isolatedhost cell comprising the vector of claim
 4. 7. A process for producing apolypeptide encoded by said polynucleotide segment comprising culturingthe host cell of claim 5 under conditions sufficient for the productionof said polypeptide.
 8. An isolated polynucleotide segment encoding amature polypeptide expressed by a polynucleotide comprising SEQ ID NO: 1in deposited strain NCIMB
 40771. 9. An isolated nucleic acid segmentcomprising a nucleotide sequence which is fully complementary to thepolynucleotide segment of claim
 8. 10. An isolated vector comprising thepolynucleotide segment of claim
 8. 11. An isolated vector comprising thenucleic acid segment of claim
 9. 12. An isolated host cell comprisingthe vector of claim
 10. 13. An isolated host cell comprising the vectorof claim
 11. 14. A process for producing the mature polypeptidecomprising culturing the host cell of claim 12 under conditionssufficient for the production of said polypeptide.